Compositions and method for treating and preventing complications of obesity

ABSTRACT

Provided herein are compositions and methods for treating, preventing, and reducing the risk of obesity and related diseases and conditions. In particular, provided herein is a composition comprising a dry powder of fruits and vegetables and uses thereof.

This application is a continuation of U.S. patent application Ser. No.16/972,066, filed Dec. 4, 2020, which claims priority to U.S.provisional patent application Ser. No. 62/681,935, filed Jun. 7, 2018,which is incorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant number58-1950-4-003 awarded by the United States Department of Agriculture.The government has certain rights in the invention.

FIELD

Provided herein are compositions and methods for treating, preventing,and reducing the risk of obesity and related diseases and conditions. Inparticular, provided herein is a composition comprising a dry powder offruits and vegetables and uses thereof.

BACKGROUND

The prevalence of obesity and metabolic disorders is rapidly increasingaround the world. In addition to metabolic disorders, obesity isassociated with coronary heart disease, type 2 diabetes, certain typesof cancer, high blood pressure, stroke, liver and gallbladder disease,sleep apnea and respiratory problems, osteoarthritis, and gynecologicalproblems in females.

Metabolic disorders are defined as dysfunction of metabolically activetissues such as adipose tissue and liver. Dysregulated immune responseas manifested by increased inflammation is one of the major contributorsto the development of metabolic disorder (Eheim et al., 2014; Sittipo etal., 2018). Adipose tissue, once viewed solely as an energy reservoir,is currently recognized as an endocrine organ that functions inregulation of metabolism (Booth et al., 2016; Galic et al., 2010). Whenthe dietary lipid load exceeds the storage capacity of dysfunctionaladipose tissue, overflow of free fatty acids will spill into circulationresulting in ectopic lipid accumulation in other tissues such as liver(Bosy-Westphal et al., 2019). Accumulation of fat in the liver canresult in non-alcoholic fatty liver disease (NAFLD). NAFLD, the leadingcause of chronic liver disease, may either be associated with(Arias-Loste et al., 2015; Polyzos et al., 2017) or be independent ofobesity (Nakamura et al., 2018; Yousef et al., 2017). In animal studies,diet-induced NAFLD was found not only in mice fed a high fat diet(typically 45% to 60% of calories from fat) but also in those fed thestandard AIN-93 diet (16% of calories from fat) (Farias Santos et al.,2015; Santos et al., 2015).

There is an urgent need for compositions for treating and preventingdisorders associated with obesity such as metabolic and cardiovasculardisorders.

SUMMARY

The present disclosure provides compositions and methods for treatingand preventing obesity and related complications. Experiments describedherein utilized a fruit and vegetable (F&V) mixture comprising 24 F&V.Supplementing mice on a Basal or high fat (HF) diet with 15% F&V mixture(w/w) (equivalent to 8-9 servings of F&V/day for humans) preventedhepatic steatosis and suppressed epididymal adipose tissue inflammation,independent of weight loss. These effects correlated with lower levelsof pro-inflammatory cytokine TNFα and ceramides, as well as increasedgut microbiota diversity and altered gut bacterial composition. Furtherexperiments demonstrated prevention of HF-induced atherosclerosis andhepatic steatosis, which may be mediated through improved dyslipidemiaand reduced inflammation.

Accordingly, provided herein is a composition, comprising: a dry powder(e.g., freeze-dried powder) comprising a mixture of fruit species and amixture of vegetables species. In some embodiments, the composition is anutritional supplement (e.g., tablet, powder, capsule, etc.), food, orbeverage.

The present disclosure is not limited to particular fruits andvegetables or components of a F&V composition. In some embodiments, thefruit species are one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,or all) fruit species selected from, for example, oranges, apples,bananas, grapes, watermelon, pineapple, strawberries, cantaloupe,lemons, grapefruit, peaches, or pears. In some embodiments, thevegetable species are one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, or all) selected from, for example, potatoes, tomatoes, sweet corn,onions, head lettuce, romaine, bell peppers, carrots, cucumbers,cabbage, beans, or sweet potato. In some embodiments, the compositioncomprises at least the recommended daily levels of fruits and vegetablesfor a particular subject (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, or more times the recommended daily levels). In some embodiments, acomposition intended to provide a daily dose comprises 40-65 grams ofdry powder. In some embodiments, the mixture of fruits and vegetablescomprises the specific F&V in the amounts listed in FIG. 10A or B. Insome embodiments, the mixture of fruit and vegetable species comprises(all percentages are w/w of the F&V mixture) at least one of 16-20%oranges, 14-18% tomatoes, 8-11% apples, 13-16% potatoes, 3.0-5.5%bananas, 3-4% sweet corn, 3-4% grapes, 2-3% lettuce, 1-2% escarole, 1-2%brussels sprouts, 1-2% cabbage, 1-2% carrots, 1-3% onions, 1-2% greenpeas, 0.5-1.5% watermelon, 0.5-1.5% honeydew melon, 0.5-1.5% broccoli,1-2% spinach, 0.5-1.5% peppers, 0.5-1.5% snap beans, 0.5-1.5%cantaloupe, 0.4-1.2% cauliflower, 0.5-1.0% mangoes, 0.5-1.0% papaya,0.3-0.9% celery, 0.4-1.2% cucumbers, 0.5-1.0% pineapple, 0.25-0.75%tangerines, 0.25-0.75% limes, 0.25-0.75% strawberries, 0.25-0.75%raspberries, 0.25-0.75% grapefruit, 0.25-0.75% lemons, 0.25-0.75%cranberries, 0.3-0.5% plums, 0.3-0.5% peaches, 0.3-0.5% cherries,0.3-0.5% blueberries, 0.3-0.5% apricots, 0.1-0.15% dried peas, 0.1-0.15%great northern beans, 0.1-0.15% dried navy beans, 0.1-0.15% driedlentils, 0.1-0.15% pinto beans, 0.1-0.15% lima beans, 0.1-0.15% redkidney beans, and 0.1-0.15% black beans (e.g., approximately 18.075%oranges, 16.161% tomatoes, 9.595% apples, 14.493% potatoes, 4.373%bananas, 3.564% sweet corn, 3.383% grapes, 2.537% lettuce, 1.651%escarole, 1.375% brussels sprouts, 1.375% cabbage, 1.329% carrots,2.017% onions, 1.293% green peas, 1.058% watermelon, 1.058% honeydewmelon, 0.84% broccoli, 1.651% spinach, 1.087% peppers, 1.061% snapbeans, 1.058% cantaloupe, 0.84% cauliflower, 0.732% mangoes, 0.732%papaya, 0.626% celery, 0.814% cucumbers, 0.732% pineapple, 0.555%tangerines, 0.555% limes, 0.437% strawberries, 0.437% raspberries,0.555% grapefruit, 0.555% lemons, 0.437% cranberries, 0.388% plums,0.388% peaches, 0.388% cherries, 0.437% blueberries, 0.388% apricots,0.121% dried peas, 0.121% great northern beans, 0.121% dried navy beans,0.121% dried lentils, 0.121% pinto beans, 0.121% lima beans, 0.121% redkidney beans, and 0.121% black beans).

In some embodiments, the F&V mixture comprises a polyphenol content of15-25% hesperetin, 15-25% caffeoylquinic acid, 10-20% quercetin, and5-15% malvidin (e.g., 20.6% hesperetin, 19.1% caffeoylquinic acid, 15.7%quercetin, and 10.3% malvidin). In some embodiments, the compositionfurther comprises 1-10% naringenin, 1-10% pelargonidin, 1-5% catechin,and 1-5% procyanidin (e.g., 6.5% naringenin, 5.8% pelargonidin, 4.2%catechin, and 3.1% procyanidin). In some embodiments, the compositionfurther comprises one or more polyphenols selected from, for example,caffeic acid, peonidin, cyanidin, pinoresinol, p-Coumaroyl, luteolin,petunidin, daidzein, genistein, ellagic acid, or gallic acid.

Additional embodiments provide a composition, comprising, consistingessentially of, or consisting of: a dry powder comprising 16-20%oranges, 14-18% tomatoes, 8-11% apples, 13-16% potatoes, 3.0-5.5%bananas, 3-4% sweet corn, 3-4% grapes, 2-3% lettuce, 1-2% escarole, 1-2%brussels sprouts, 1-2% cabbage, 1-2% carrots, 1-3% onions, 1-2% greenpeas, 0.5-1.5% watermelon, 0.5-1.5% honeydew melon, 0.5-1.5% broccoli,1-2% spinach, 0.5-1.5% peppers, 0.5-1.5% snap beans, 0.5-1.5%cantaloupe, 0.4-1.2% cauliflower, 0.5-1.0% mangoes, 0.5-1.0% papaya,0.3-0.9% celery, 0.4-1.2% cucumbers, 0.5-1.0% pineapple, 0.25-0.75%tangerines, 0.25-0.75% limes, 0.25-0.75% strawberries, 0.25-0.75%raspberries, 0.25-0.75% grapefruit, 0.25-0.75% lemons, 0.25-0.75%cranberries, 0.3-0.5% plums, 0.3-0.5% peaches, 0.3-0.5% cherries,0.3-0.5% blueberries, 0.3-0.5% apricots, 0.1-0.15% dried peas, 0.1-0.15%great northern beans, 0.1-0.15% dried navy beans, 0.1-0.15% driedlentils, 0.1-0.15% pinto beans, 0.1-0.15% lima beans, 0.1-0.15% redkidney beans, and 0.1-0.15% black beans.

Certain embodiments provide a food or beverage product comprising acomposition as described herein. In some embodiments, the productcomprises 2%-20% (e.g., 5-15%, 5-20%, or 5-10%) (w/w) of thecomposition. In some embodiments, the food or beverage product comprisesone or more of protein, carbohydrates, and fat (e.g., 5-15% protein(kcal/kcal), 75-85% carbohydrates (kcal/kcal), and 0-20% fats(kcal/kcal); 10-12% protein (kcal/kcal), 80-83% carbohydrates(kcal/kcal), and 5-10% fat (kcal/kcal), or 11.6% protein (kcal/kcal),81.2% carbohydrates, and 7.2% fats (kcal/kcal).

Further embodiments provide a nutritional supplement comprising acomposition as described herein.

Yet other embodiments provide a composition as described herein for usein a method of treating and/or preventing one or more conditionsselected from, for example, weight gain, obesity, inflammatoryconditions, fatty liver disease, high cholesterol, glucose intolerance,insulin resistance, low gut microbiota diversity, heart disease, oratherosclerosis.

Still other embodiments provide a composition as described herein foruse in a method of any one or more of the following: decreasing fatmass, increasing muscle mass, reducing inflammatory cytokines and/orceramides, reducing tissue inflammation, decreasing cholesterol,improving glucose tolerance, improving immune response, increasing gutmicrobiota diversity, increasing lifespan, improving cognition, orimproving bone health.

Also provided herein is the use of a composition as described herein fortreating and/or preventing one or more conditions selected from weightgain, obesity, inflammatory conditions, fatty liver disease, highcholesterol, glucose intolerance, insulin resistance, low gut microbiotadiversity, heart disease, and atherosclerosis.

Further provided herein is the use of a composition as described hereinin decreasing fat mass, increasing muscle mass, reducing inflammatorycytokines and/or ceramides, reducing tissue inflammation, decreasingcholesterol, improving glucose tolerance, treating or preventingatherosclerosis, improving immune response, increasing gut microbiotadiversity, increasing lifespan, improving cognition, and/or improvingbone health.

Still further embodiments provide a method of treating and/or preventingone or more conditions selected from, for example, weight gain, obesity,inflammatory conditions, fatty liver disease, high cholesterol, glucoseintolerance, insulin resistance, low gut microbiota diversity, heartdisease, and atherosclerosis in a subject, comprising: administering acomposition as described herein to the subject.

Embodiments of the disclosure provide a method of decreasing fat mass,increasing muscle mass, reducing inflammatory cytokines and/orceramides, reducing tissue inflammation, decreasing cholesterol,improving glucose tolerance, improving immune response, increasing gutmicrobiota diversity, increasing lifespan, improving cognition, and/orimproving bone health in a subject, comprising: administering acomposition as described herein to the subject.

In some embodiments, the subject is a human. In some embodiments, thesubject exhibits one or more signs or symptoms of the condition and theadministering reduces or eliminates the signs or symptoms. In someembodiments, the administering comprises administering at least a dailydose equivalent to at least 5 servings of F&V for the subject (e.g.,40-65 grams of dry powder per day). In some embodiments, theadministering is repeated one or more (e.g., 1, 2, 3, or more) times perday for a period of at least 1 week (e.g., at least 1 month, at leastone year, multiple years, or indefinitely).

Additional embodiments are described herein.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that mice fed HF diet with F&V supplementation had lessbody weight gain and fat tissue mass. Six-week old male C57BL/6J micewere fed a basal diet (B, 16 kcal % fat) or high fat diet (HF, 45 kcal %fat) with 0%, 5%, 10%, or 15% F&V supplementation, respectively, for 20weeks. Mice fed a matched control diet (MC, 10 kcal % fat) were used ascontrol for HF diet. (A, B) Body weight; (C, D, E, F) Fat tissue; (G, H,I, J) Lean tissue. Data are presented as mean±SEM. # P<0.05, ###P<0.001, and N.S. not significant.

FIG. 2 shows that F&V supplementation suppressed inflammatory cellinfiltration in adipose tissue and prevented NAFLD. Representativeimages of eAT sections stained with H&E (A, B, D, E, and G) are shown.Adipocyte size distribution of eAT in the mice fed basal diet with 0% or15% F&V (2C) and high fat diet with 0% or 15% F&V as well as matchedcontrol diet (2F) are shown. The pictures (H-I, K-L, and N) arerepresentative images of H/E stained liver tissue sections. Hepaticsteatosis area is expressed as mean±SE. (0) Table of hepatic steatosiscorrelation with serum ceramides and TNFα. # P<0.05, ## P<0.01, ###P<0.001. N.S. not significant.

FIG. 3 shows that F&V supplementation reduces serum and liver ceramidelevels. (A-X) Serum and liver levels of total ceramides and indicatedceramide species were determined. # P<0.05, ## P<0.01, N.S. notsignificant.

FIG. 4 shows effects of F&V supplementation on the levels of liver FXRexpression, nSMase activity, and levels of systemic and liver TNFα. Thelevels of serum and liver pro-inflammatory cytokine TNFα (4A, 4E, 4C,and 4G) were measured by ELISA. The mRNA levels of liver FXR (4B and 4F)were quantitated by RTqPCR. Hepatic nSMase activity (4D-4H) weredetermined using C6-NBD-SM as a substrate, and generated fluorescentproduct, NBD-ceramide, was monitored by a reverse phase HPLC.

FIG. 5 shows that F&V supplementation in either basal or HF dietincreased gut bacterial diversity. A and C: F&V treatment increasedalpha-diversity in basal (A) and HF diet (B). B and D: PrincipalCoordinate Analysis plot with unweighted unifrac distance showsdifferential clustering of the three diet groups. # P<0.05; ### P<0.001.N.S. no significant.

FIG. 6 shows that F&V supplementation changed bacterial communitycomposition. Gut bacterial composition in different diets are shown inphylum level (A), class level (B), order level (C), family level (D) andgenus level (E).

FIG. 7 shows a correlation matrix heatmap based on Spearman correlationcoefficients comparing two diet feeding groups. (A). mice fed Basal dietwith 0% or 15% F&V. (B). HF diet with 0% or 15% F&V. Star symbolindicates significant correlation after correcting for multiple testingusing the Benjamini-Hochberg method.

FIG. 8 shows that mice fed HFD+15% F&V had lower spleen weight andspleen weight index.

FIG. 9 shows FIG. 27 shows lipidomic changes of lipoxin, 14, 15-EET,20-HETE, and DHGLA in mice fed a diet with 15% F&V.

FIG. 10A-C shows exemplary compositions of the fruit and vegetablecomponents of animal diets.

FIG. 11 shows that F&V supplementation increased fecal weight, fecalenergy density, and fecal energy excretion (A-J). # P<0.05, ## P<0.01.N.S. no significant.

FIG. 12 shows that F&V supplementation down-regulated pro-inflammatorymRNA expression in eAT. (A-D) The levels of pro-inflammatory mRNAexpression in eAT were quantitated by RTqPCR. # P<0.05. N.S. nosignificant. IL-10: interleukin 10; IL-6: IL-6: interleukin 6; MCP-1:monocyte chemoattractant protein-1; TNFα: tumor necrosis factor α.

FIG. 13 shows that F&V supplementation had no significant effects onserum lipids profiling. Serum total cholesterol (A, B), LDL levels (C,D), HDL levels (E, F), VLDL levels (G, H), TG levels (I, J),serum-non-HDL and non/HDL/HDL ratio (K) are expressed as mean±SE. #P<0.05, ## P<0.01, ### P<0.001. N.S. no significant.

FIG. 14 shows that F&V supplementation did not significant affect liverceramide synthases mRNA expression. (A-C) The mRNA levels of liverceramide synthases were quantitated by RTqPCR. # P<0.05, N.S. notsignificant.

FIG. 15 shows that F&V supplementation significantly changed abundanceof specific bacterial community composition. (A-D) Differentialabundance of gut bacteria between groups was analyzed using Deseq2package.

FIG. 16 shows that F&V supplementation improved glucose tolerance anddiet-induced suppression of T cell proliferation and T cell expansion.(A-D) Intraperitoneal glucose tolerance test (IPGTT) was performed. (E)³H-thymidine incorporation and % of CD4 and CD8 T cells.

FIG. 17 shows that F&V supplementation suppressed high fat diet-inducedaortic atherosclerosis in LDLR KO mouse. Four-week old male LDL receptorknockout mice were fed a low fat diet (LF, 10% calories from fat; 52 mgcholesterol/1000 kcal), or high fat diet (HF, 27% calories from fat; 130mg cholesterol/1000 kcal) or HF diet with 15% F&V supplementation(HF15), respectively, for 20 weeks. (A_Representative images of aortasstained en face with Oil Red O. (B) the ratios of the plaque areastained with oil red O over total aorta area were quantitated. ###P<0.001.

FIG. 18 shows effects of F&V supplementation on body weight and liverweight in LDLR KO mouse. (A-C) Liver weight was measured and percentageof liver weight to body was calculated. ## P<0.01, ### P<0.001, N.S. nosignificance.

FIG. 19 shows that F&V supplementation prevented high fat diet-inducedhepatic steatosis in LDLR KO mouse. (A), HF0 diet (B), and HF15 diet(C). D, Quantitation of hepatic steatosis area of three diet groups. ###P<0.001.

FIG. 20 shows that F&V supplementation improved dyslipidemia in LDLR KOmouse. Plasma lipids profile (A-E) was determined. Ratio of TG/HDL (F),LDL/HDL (G), non HDL/HDL (H), and plasma VLDL (I) were calculated. #P<0.05, ## P<0.01, ### P<0.001. N.S. no significant.

FIG. 21 shows effects of F&V supplementation on plasma pro-inflammatorycytokine levels in LDLR KO mouse. (A-D) Serum cytokine levels weredetermined by using the Meso Scale Discovery (MSD) multiplex ELISAplatform. # P<0.05, ## P<0.01, ### P<0.001. N.S. no significant.

FIG. 22 shows that F&V supplementation reduced liver mRNA levels ofcytokines in LDLR KO mouse. (A-D) Liver mRNA levels of Fasn, IL-6,PPARg, SREBF land TNFα were quantitated using RTqPCR. # P<0.05.

FIG. 23 shows a heatmap of Spearman's correlations shows aorticatherosclerotic lesion and hepatic steatosis area vs plasma TNFα andlipid profile in LDLR KO mouse. # P<0.05, ## P<0.01, ### P<0.001.

FIG. 24 shows that F&V supplementation in HF diet increased gutbacterial diversity. (A) F&V treatment increased alpha-diversity. (B)Principal Coordinate Analysis plot with unweighted unifrac distanceshows differential clustering of the HF15 diet group from LF diet groupand HF0 group. ## P<0.01. N.S. no significant.

FIG. 25 shows that F&V supplementation changed bacterial communitycomposition. Gut bacterial composition in different diets are shown inphylum level (A), class level (B), order level (C), family level (D) andgenus level (E).

FIG. 26 shows that F&V supplementation changed bacterial communitycomposition. Gut bacterial composition in different diets are shown inphylum level (A), class level (B), order level (C), family level (D) andgenus level (E).

FIG. 27 shows that F&V supplementation improved dyslipidemia in LDLR KOmouse. (A-D) Plasma lipid profiles of (A) cholesterol, (B)triglycerides, (C) HDL, and (E) LDL.

FIG. 28 shows that F&V supplementation reduced or had no impact onplasma cytokine levels in LDLR KO mouse. (A-H) Serum cytokine levelswere determined by using the Meso Scale Discovery (MSD) multiplex ELISAplatform.

DEFINITIONS

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of embodimentsdescribed herein, some preferred methods, compositions, and materialsare described herein. However, before the present materials and methodsare described, it is to be understood that this invention is not limitedto the particular molecules, compositions, methodologies or protocolsherein described, as these may vary in accordance with routineexperimentation and optimization. It is also to be understood that theterminology used in the description is for the purpose of describing theparticular versions or embodiments only and is not intended to limit thescope of the embodiments described herein.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. However, in case of conflict,the present specification, including definitions, will control.Accordingly, in the context of the embodiments described herein, thefollowing definitions apply.

As used herein and in the appended claims, the singular forms “a”, “an”and “the” include plural reference unless the context clearly dictatesotherwise.

As used herein, the term “about,” when referring to a value or to anamount of mass, weight, time, volume, concentration or percentage ismeant to encompass variations of in some embodiments ±20%, in someembodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, insome embodiments ±0.5%, and in some embodiments ±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethod. As used herein, the terms “comprise”, “include”, and linguisticvariations thereof denote the presence of recited feature(s),element(s), method step(s), etc. without the exclusion of the presenceof additional feature(s), element(s), method step(s), etc. Conversely,the term “consisting of” and linguistic variations thereof, denotes thepresence of recited feature(s), element(s), method step(s), etc. andexcludes any unrecited feature(s), element(s), method step(s), etc.,except for ordinarily-associated impurities. The phrase “consistingessentially of” denotes the recited feature(s), element(s), methodstep(s), etc. and any additional feature(s), element(s), method step(s),etc. that do not materially affect the basic nature of the composition,system, or method. Many embodiments herein are described using open“comprising” language. Such embodiments encompass multiple closed“consisting of” and/or “consisting essentially of” embodiments, whichmay alternatively be claimed or described using such language.

As used herein, the terms “co-administration” and variations thereofrefer to the administration of at least two agent(s) or therapies to asubject (e.g., a composition disclosed herein and one or moretherapeutic agents). In some embodiments, the co-administration of twoor more agents or therapies is concurrent. In other embodiments, a firstagent/therapy is administered prior to a second agent/therapy. Those ofskill in the art understand that the formulations and/or routes ofadministration of the various agents or therapies used may vary. Theappropriate dosage for co-administration can be readily determined byone skilled in the art. In some embodiments, when agents or therapiesare co-administered, the respective agents or therapies are administeredat lower dosages than appropriate for their administration alone. Thus,co-administration is especially desirable in embodiments where theco-administration of two or more agents results in sensitization of asubject to beneficial effects of one of the agents via co-administrationof the other agent.

As used herein, the term “pharmaceutical composition” refers to thecombination of an active agent with a carrier, inert or active, makingthe composition especially suitable for therapeutic use.

The terms “pharmaceutically acceptable” or “pharmacologicallyacceptable”, as used herein, refer to compositions that do notsubstantially produce adverse reactions, e.g., toxic, allergic, orimmunological reactions, when administered to a subject.

As used herein, the terms “prevent,” “prevention,” and preventing” mayrefer to reducing the likelihood of a particular condition or diseasestate (e.g., metabolic disorder or other complication of obesity) fromoccurring in a subject not presently experiencing or afflicted with thecondition or disease state. The terms do not necessarily indicatecomplete or absolute prevention. For example “preventing metabolicdisorder” refers to reducing the likelihood of metabolic disorder andrelated conditions occurring in a subject not presently experiencing ordiagnosed with metabolic disorder. The terms may also refer to delayingthe onset of a particular condition or disease state (e.g., metabolicdisorder) in a subject not presently experiencing or afflicted with thecondition or disease state. In order to “prevent metabolic disorder” acomposition or method need only reduce the likelihood and/or delay theonset of metabolic disorder or related condition, not completely blockany possibility thereof. “Prevention,” encompasses any administration orapplication of a therapeutic or technique to reduce the likelihood ordelay the onset of a disease developing (e.g., in a mammal, including ahuman). Such a likelihood may be assessed for a population or for anindividual.

As used herein, the terms “treat,” “treatment,” and “treating” refer toreducing the amount or severity of a particular condition, disease state(e.g., metabolic disorders or other complications of obesity), orsymptoms thereof, in a subject presently experiencing or afflicted withthe condition or disease state. The terms do not necessarily indicatecomplete treatment (e.g., total elimination of the condition, disease,or symptoms thereof). “Treatment,” encompasses any administration orapplication of a therapeutic or technique for a disease (e.g., in amammal, including a human), and includes inhibiting the disease,arresting its development, relieving the disease, causing regression, orrestoring or repairing a lost, missing, or defective function; orstimulating an inefficient process.

As used herein, the term w/w (weight/weight) refers to the amount of agiven substance in a composition on weight basis. For example, acomposition comprising 50% w/w carrots means that the mass of thecarrots is 50% of the total mass of the composition (i.e., 50 grams ofcarrots in 100 grams of the composition, such as a mixture of F&V).

As used herein, the terms “food” and “food products” refer to productsand ingredients therefore, taken by the mouth, the constituents of whichare active in and/or absorbed by the G.I. tract with the purpose ofnourishment of the body and its tissues, refreshment and indulgence,which products are to be marketed and sold to customers for consumptionby humans. Examples of foods and food and beverage products include, butare not limited to, tea; spreads; ice cream; frozen fruits andvegetables; snacks including diet foods and beverages; condiments; andculinary aids. In some embodiments, a “food” is a material comprisingprotein, carbohydrate and/or fat, which is used in the body of anorganism to sustain growth, repair and vital processes and to furnishenergy. Foods may also contain supplementary substances such asminerals, vitamins and condiments. See Merriam-Webster's CollegiateDictionary, 10th Edition, 1993.

As used herein a “food additive” (e.g., as defined by the FDA in 21C.P.R. 170.3 (e)(1)) includes direct and indirect additives.

As used herein, a “dietary supplement” is a product that is intended tosupplement a diet. In some embodiments, dietary supplements containlittle or no calories.

As used herein, the term “subject” as used herein includes all membersof the animal kingdom including mammals, and suitably refers to humans.

DETAILED DESCRIPTION

Obesity-induced alteration in adipose tissue, especially visceral whiteadipose tissue, is characterized by infiltrated macrophages and otherinflammatory cells, release of cytokines and chemokines such as monocytechemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), and tumornecrosis factor-alpha (TNF-α), increased lipolysis, and death ofadipocytes, all of which play a critical role in pathogenesis of NAFLD(Cheng et al., 2015; Wei et al., 2018). In particular, TNFα has beenfound to play a pivotal role in the development and progression of NAFLDin murine models. High circulating levels of TNFα is associated with theseverity of NAFLD in morbidly obese patients (Kakino et al., 2018;Paredes-Turrubiarte et al., 2016). TNFα stimulates liver ceramidegeneration (Dbaibo et al., 2001; Engin, 2017), a unique class ofsphingolipid signaling lipid molecules that are involved in pathogenesisof NAFLD (Nikolova-Karakashian, 2018), indicating that TNFα andceramides may additively or synergistically promote NAFLD. Ceramide canbe formed by de novo synthesis pathway or through the salvage pathwayusing ceramide synthase. It may also be produced from sphingomyelin viathe function of enzyme sphingomyelinase, the activity of which isincreased by oxidative stress and TNFα.

The farnesoid X receptor (FXR), a nuclear receptor abundantly expressedin liver, is a key regulator controlling various hepatic metabolicprocesses. Hepatic FXR activation inhibits the expression ofpro-inflammatory genes, including that of TNFα, by blocking NFκBactivation (Kim et al., 2015; Wang et al., 2008). Further, hepatic FXRexpression levels were lower in NAFLD patients (Yang et al., 2010) anddiet-induced mice NAFLD (Nie et al., 2017). FXR is a potential drugtarget for treatment of NAFLD (Ali et al., 2015; Li et al., 2013).

Gut microbial dysbiosis, characterized by low diversity and alteredcomposition of gut microbiota, has been shown to be associated withobesity and metabolic disorders in humans (Qin et al., 2012; Turnbaughet al., 2009) and causally related to these disorders in rodent models(Kriss et al., 2018; Li et al., 2017; Ridaura et al., 2013).Furthermore, gut microbiota dysbiosis may contribute to NAFLDpathogenesis (Bibbo et al., 2018; Saltzman et al., 2018; Wieland et al.,2015).

Dietary patterns affect gut microbiota, oxidative stress, inflammation,and metabolism (Kong et al., 2014; Sheflin et al., 2017; Tindall et al.,2018; Wong, 2014). Experiments described herein determined the impact ofincreased consumption of F&V on metabolic disorders and its underlyingmechanisms. Results indicated that high intake of a variety of F&Vcompletely prevented metabolic dysfunction of adipose tissue and NAFLDindependent of body weight reduction. Furthermore, these data show thatthese effects of F&V on metabolic disorders are associated withincreased gut microbiota diversity and reduction in pro-inflammatorycytokine and ceramide levels.

Further experiments demonstrated that mice fed HF diet had significantlyhigher plasma TG and LDL and lower HDL levels than mice fed LF diet, andthis dyslipidemia was prevented by F&V supplementation. Further, theHF+FV group had lower plasma TNFα levels compared to HF0 group (p<0.05).Spearman correlation analysis showed that aortic atherosclerotic lesionand hepatic steatosis area were negatively correlated with plasma HDL(p<0.001) and significantly and positively correlated with TNFα, and theratios of LDL/HDL, TG/HDL, and non HDL/HDL.

Accordingly, in some embodiments, provided herein is a formulation ofF&V comprising, for example, a freeze-dried mixture of 12 fruits and 12vegetables (e.g., as described in FIG. 10A-C) and methods of using suchcompositions to treat, prevent, and reduce the risk of signs, symptoms,and complications related to metabolic disorder, obesity, and relateddisorders.

The present disclosure is not limited to particular fruits andvegetables or components of a F&V composition. In some embodiments, thefruit species are one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,or all) fruit species selected from, for example, oranges, apples,bananas, grapes, watermelon, pineapple, strawberries, cantaloupe,lemons, grapefruit, peaches, or pears. In some embodiments, thevegetable species are one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, or all) selected from, for example, potatoes, tomatoes, sweet corn,onions, head lettuce, romaine, bell peppers, carrots, cucumbers,cabbage, beans, or sweet potato.

In some embodiments, the mixture of fruit and vegetable speciescomprises, consists essentially of, or consists of at least one of16-20% oranges, 14-18% tomatoes, 8-11% apples, 13-16% potatoes, 3.0-5.5%bananas, 3-4% sweet corn, 3-4% grapes, 2-3% lettuce, 1-2% escarole, 1-2%brussels sprouts, 1-2% cabbage, 1-2% carrots, 1-3% onions, 1-2% greenpeas, 0.5-1.5% watermelon, 0.5-1.5% honeydew melon, 0.5-1.5% broccoli,1-2% spinach, 0.5-1.5% peppers, 0.5-1.5% snap beans, 0.5-1.5%cantaloupe, 0.4-1.2% cauliflower, 0.5-1.0% mangoes, 0.5-1.0% papaya,0.3-0.9% celery, 0.4-1.2% cucumbers, 0.5-1.0% pineapple, 0.25-0.75%tangerines, 0.25-0.75% limes, 0.25-0.75% strawberries, 0.25-0.75%raspberries, 0.25-0.75% grapefruit, 0.25-0.75% lemons, 0.25-0.75%cranberries, 0.3-0.5% plums, 0.3-0.5% peaches, 0.3-0.5% cherries,0.3-0.5% blueberries, 0.3-0.5% apricots, 0.1-0.15% dried peas, 0.1-0.15%great northern beans, 0.1-0.15% dried navy beans, 0.1-0.15% driedlentils, 0.1-0.15% pinto beans, 0.1-0.15% lima beans, 0.1-0.15% redkidney beans, and 0.1-0.15% black beans (e.g., approximately 18.075%oranges, 16.161% tomatoes, 9.595% apples, 14.493% potatoes, 4.373%bananas, 3.564% sweet corn, 3.383% grapes, 2.537% lettuce, 1.651%escarole, 1.375% brussels sprouts, 1.375% cabbage, 1.329% carrots,2.017% onions, 1.293% green peas, 1.058% watermelon, 1.058% honeydewmelon, 0.84% broccoli, 1.651% spinach, 1.087% peppers, 1.061% snapbeans, 1.058% cantaloupe, 0.84% cauliflower, 0.732% mangoes, 0.732%papaya, 0.626% celery, 0.814% cucumbers, 0.732% pineapple, 0.555%tangerines, 0.555% limes, 0.437% strawberries, 0.437% raspberries,0.555% grapefruit, 0.555% lemons, 0.437% cranberries, 0.388% plums,0.388% peaches, 0.388% cherries, 0.437% blueberries, 0.388% apricots,0.121% dried peas, 0.121% great northern beans, 0.121% dried navy beans,0.121% dried lentils, 0.121% pinto beans, 0.121% lima beans, 0.121% redkidney beans, and 0.121% black beans). All percentages are w/w % of theF&V mixture.

In some embodiments, composition provide at least 5 (e.g., at least 5,6, 7, 8, 9, 10, 15 or more) of the recommended daily amounts (e.g.,servings) of F&V for an average adult (See e.g.,health.gov/dietaryguidelines/dga2000/document/build.htm; hereinincorporated by reference in its entirety). For example, in someembodiments, a composition intended to be a single daily dose for anaverage adult comprises 40-65 g of dry powder (e.g., plus or minus 1, 5,10, 15, or 20%). The amounts are adjusted for children or individualswith specific dietary need.

In some embodiments, the composition comprises a plurality ofpolyphenols and other beneficial molecules or compounds. For example, insome embodiments, the F&V mixture comprises a polyphenol content of15-25% hesperetin, 15-25% caffeoylquinic acid, 10-20% quercetin, and5-15% malvidin (e.g., 20.6% hesperetin, 19.1% caffeoylquinic acid, 15.7%quercetin, and 10.3% malvidin). In some embodiments, the compositionfurther comprises 1-10% naringenin, 1-10% pelargonidin, 1-5% catechin,and 1-5% procyanidin (e.g., 6.5% naringenin, 5.8% pelargonidin, 4.2%catechin, and 3.1% procyanidin). All percentages are w/w % of the F&Vmixture. In some embodiments, the composition further comprises one ormore polyphenols selected from, for example, caffeic acid, peonidin,cyanidin, pinoresinol, p-Coumaroyl, luteolin, petunidin, daidzein,genistein, ellagic acid, or gallic acid.

In some embodiments, the composition is provided as a dry powder. Insome embodiments, the powder is provided as a freeze-dried powder (e.g.,prepared as described in Example 1).

The powder is used in different forms including but not limited toencapsulated, added to liquid consumables, dairy and dairy substituteproducts, bars, and sashes. It may also be printed using 3-D printing,to create products with different shapes and consistency.

In some embodiments, the composition is provided as a powder and theuser adds the powder to a beverage or food. In some embodiments, thecomposition is provided as a ready to eat beverage or food product.

In some embodiments, the food or beverage product comprises 2%-20%(e.g., 5-15%, 5-20%, or 5-10%) (w/w) of the F&V composition.

In some embodiments, the beverage, nutritional supplement, or foodproduct comprises 0-60% protein (kcal/kcal), 0-99% carbohydrates(kcal/kcal), and 0-60% fats (kcal/kcal) (e.g., 5-15% protein(kcal/kcal), 75-85% carbohydrates (kcal/kcal), and 0-20% fats(kcal/kcal); 10-12% protein (kcal/kcal), 80-83% carbohydrates(kcal/kcal), and 5-10% fat (kcal/kcal), or 11.6% protein (kcal/kcal),81.2% carbohydrates, and 7.2% fats (kcal/kcal) (percentages of the totalcomposition),

In some embodiments, the composition is provided as a nutritionalsupplement or pharmaceutical formulation for oral delivery.

In some embodiments, the present disclosure provides a supplementcomposition comprising one or more of the foregoing compositions incombination with a pharmaceutically acceptable carrier. The actual formof the carrier, and thus, the composition itself, is not critical. Thecarrier may be a liquid, gel, gelcap, capsule, powder, solid tablet(coated caplet or non-coated), tea, or the like. The composition, inthis case, is preferably in the form of a tablet or capsule and mostpreferably in the form of a soft gel capsule. Suitable excipient and/orcarriers include maltodextrin, calcium carbonate, dicalcium phosphate,tricalcium phosphate, microcrystalline cellulose, dextrose, rice flour,magnesium stearate, stearic acid, croscarmellose sodium, sodium starchglycolate, crospovidone, sucrose, vegetable gums, lactose,methylcellulose, povidone, carboxymethylcellulose, corn starch, and thelike (including mixtures thereof). Preferred carriers include calciumcarbonate, magnesium stearate, maltodextrin, and mixtures thereof. Thevarious ingredients and the excipient and/or carrier are mixed andformed into the desired form using conventional techniques. The tabletor capsule may be coated with an enteric coating that dissolves at a pHof about 6.0 to 7.0. A suitable enteric coating that dissolves in thesmall intestine but not in the stomach is cellulose acetate phthalate.Further details on techniques for formulation for and administration maybe found in the latest edition of Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.).

The dietary supplement may comprise one or more inert ingredients,especially if it is desirable to limit the number of calories added tothe diet by the dietary supplement. For example, the dietary supplementmay also contain optional ingredients including, for example, herbs,vitamins, minerals, enhancers, colorants, sweeteners, flavorants, inertingredients, and the like.

In further embodiments, the compositions comprise at least one foodflavoring such as acetaldehyde, acetoin (acetyl methylcarbinol),anethole (parapropenyl anisole), benzaldehyde (benzoic aldehyde), Nbutyric acid (butanoic acid), d or l carvone (carvol), cinnamaldehyde(cinnamic aldehyde), citral (2,6 dimethyloctadien 2,6 al 8, gera nial,neral), decanal (N decylaldehyde, capraldehyde, capric aldehyde,caprinaldehyde, aldehyde C 10), ethyl acetate, ethyl butyrate, 3 methyl3 phenyl glycidic acid ethyl ester (ethyl methyl phenyl glycidate,strawberry aldehyde, C 16 aldehyde), ethyl vanillin, geraniol (3,7dimethyl 2,6 and 3,6 octadien 1 ol), geranyl acetate (geraniol acetate),limonene (d, l, and dl), linalool (linalol, 3,7 dimethyl 1,6 octadien 3ol), linalyl acetate (bergamol), methyl anthranilate (methyl 2aminobenzoate), piperonal (3,4 methylenedioxy benzaldehyde,heliotropin), vanillin, alfalfa (Medicago sativa L.), allspice (Pimentaofficinalis), ambrette seed (Hibiscus abelmoschus), angelic (Angelicaarchangelica), Angostura (Galipea officinalis), anise (Pimpinellaanisum), star anise (Illicium verum), balm (Melissa officinalis), basil(Ocimum basilicum), bay (Laurus nobilis), calendula (Calendulaofficinalis), (Anthemis nobilis), capsicum (Capsicum frutescens),caraway (Carum carvi), cardamom (Elettaria cardamomum), cassia,(Cinnamomum cassia), cayenne pepper (Capsicum frutescens), Celery seed(Apium graveolens), chervil (Anthriscus cerefolium), chives (Alliumschoenoprasum), coriander (Coriandrum sativum), cumin (Cuminum cyminum),elder flowers (Sambucus canadensis), fennel (Foeniculum vulgare),fenugreek (Trigonella foenum graecum), ginger (Zingiber officinale),horehound (Marrubium vulgare), horseradish (Armoracia lapathifolia),hyssop (Hyssopus officinalis), lavender (Lavandula officinalis), mace(Myristica fragrans), marjoram (Majorana hortensis), mustard (Brassicanigra, Brassica juncea, Brassica hirta), nutmeg (Myristica fragrans),paprika (Capsicum annuum), black pepper (Piper nigrum), peppermint(Mentha piperita), poppy seed (Papayer somniferum), rosemary (Rosmarinusofficinalis), saffron (Crocus sativus), sage (Salvia officinalis),savory (Satureia hortensis, Satureia montana), sesame (Sesamum indicum),spearmint (Mentha spicata), tarragon (Artemisia dracunculus), thyme(Thymus vulgaris, Thymus serpyllum), turmeric (Curcuma longa), vanilla(Vanilla planifolia), zedoary (Curcuma zedoaria), sucrose, glucose,saccharin, sorbitol, mannitol, aspartame. Other suitable flavoring aredisclosed in such references as Remington's Pharmaceutical Sciences,18th Edition, Mack Publishing, p. 1288-1300 (1990), and Furia andPellanca, Fenaroli's Handbook of Flavor Ingredients, The Chemical RubberCompany, Cleveland, Ohio, (1971), known to those skilled in the art.

In other embodiments, the compositions comprise at least one syntheticor natural food coloring (e.g., annatto extract, astaxanthin, beetpowder, ultramarine blue, canthaxanthin, caramel, carotenal, betacarotene, carmine, toasted cottonseed flour, ferrous gluconate, ferrouslactate, grape color extract, grape skin extract, iron oxide, fruitjuice, vegetable juice, dried algae meal, tagetes meal, carrot oil, cornendosperm oil, paprika, paprika oleoresin, riboflavin, saffron, tumeric,tumeric and oleoresin).

Preferred unit dosage formulations are those containing a daily dose orunit, daily subdose, as herein above-recited, or an appropriate fractionthereof, of an agent. In some embodiments, the unit dose comprises atleast the recommended daily levels of fruits and vegetables for aparticular subject (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, ormore times the recommended daily numbers of servings), provided in oneor more doses.

The daily requirements for a particular subject vary based on age,gender, etc. (See e.g.,health.gov/dietaryguidelines/dga2000/document/build.htm). For example,the number of servings of vegetable servings recommended per day variesfrom 3-5 and the number of servings of fruit varies from 2-4. In someembodiments, a serving is equivalent to 1 cup of raw leafy vegetables, ½cup of other vegetables cooked or raw, ¾ cup of vegetable juice, 1medium apple, banana, orange, pear, ½ cup of chopped, cooked, or cannedfruit, or ¾ cup of fruit juice. In some embodiments, the compositionsdescribed herein are provided as a daily or unit dose of 20-80 (e.g.,30-70, 30-60, or 40-65 g/day) of a F&V mixture (e.g., 8-9 servings ofF&V). The amount is adjusted based on the dietary needs of theparticular subject.

It also is intended that the compositions and methods of this disclosurebe combined with other suitable compositions and therapies. For example,the compositions described herein may co-administered with one or moreadditional agents suitable for the treating and preventing metabolicdisorders and other complications of obesity.

The F&V compositions described herein find use in the treating,prevention, and reduction of risk of a number of different conditionsassociated with obesity and metabolic disorders. Examples include, butare not limited to, weight gain, obesity, inflammatory conditions, fattyliver disease, high cholesterol, glucose intolerance, insulinresistance, low gut microbiota diversity, heart disease, andatherosclerosis. In some embodiments, the compositions find use indecreasing fat mass, increasing muscle mass, reducing inflammatorycytokines and/or ceramides, reducing tissue inflammation, decreasingcholesterol, improving glucose tolerance, improving immune response,increasing gut microbiota diversity, increasing lifespan, improvingcognition, and/or improving bone health in a subject.

In some embodiments, the F&V compositions described herein areadministered to a subject diagnosed with or at risk of a disorder orcondition described herein or otherwise in need. In some embodiments,the subject is obese or not obese. In some embodiments, the subject hasone or more risk factors for a disease or condition described herein(e.g., family history, excess weight, etc.). In some embodiments, thesubject does not consume the recommended daily levels of F&V on aregular basis.

In some embodiments, the administering reduces a measure (e.g., sign,symptom, lab result) or a disorder, condition, or complication describedherein. In some embodiments, the administering prevent or reduces therisk of developing such signs, symptoms, or lab results in an individualthat does not exhibit the signs or symptoms.

In some embodiments, the administering is repeated one or more (e.g., 1,2, 3, or more) times per day for a period of at least 1 week (e.g., atleast 1 month, at least one year, multiple years, or indefinitely).

EXPERIMENTAL Example 1 A Mixture of Fruits & Vegetables PreventsDiet-Induced Hepatic Steatosis in Mice Methods Animals and Diets

Diet compositions are shown in Tables 1-5. Four-week-old C57BL/6J malemice were purchased from The Jackson Laboratory (Bar Harbor, Me., USA)and housed at the animal care facility at Jean Mayer USDA HumanNutrition Research Center on Aging at Tufts University. After 12 days ofacclimation, individually caged animals were assigned intoweight-matched 9 groups, including standard mice diet [Basal (B0), 16kcal % fat, AIN-93G; Research Diets, #D10012G), high-fat diet (HF0, 45kcal % fat, Research Diets, #D12451M), low-fat match control for HF diet(MC, 10.0 kcal % fat, Research Diets, #D12450H), and B0 and HF0supplemented with 5%, 10%, and 15% of a fruit and vegetable (F&V)mixture (see below, respectively. The nutrient content of animal dietwith or without F&V powder is presented in Table 3. Mice were fed adlibitum with respective diets for 20-weeks, during and at which timedifferent outcome variables, including body weight, food intake, andbody composition were recorded and analyzed.

Freeze-Dried Fruits and Vegetables (F&V) Powder Preparation

F&V mixture containing a combination of 24 of the most commonly consumedF&V based on USDA census data was homogenized to prepare the freezedried powder (See Tables 10A-B for F&V compositions). To mimic theAmerican patterns of consumption, the proportion of each individualfruit or vegetable in the F&V mixture was calculated based on therelative proportion of yearly F&V consumption in United States. Thefresh fruits and vegetables were purchased from grocery stores, and thenwashed and cut to get only edible part. Next, they were blended in 30000rpm blender, 15 to 20 second each time, total 2 minutes, to prepare afruit and vegetable homogenate. The F&V homogenate was saved instainless steel container, wrapped with aluminum foil and frozen in −80°C. freezer overnight. Then, the frozen F&V mixture was put into afreeze-dryer chamber (VirTis Freezemobile 35XL Freeze Dryer) with vacuumset to 300 mTorr and shelf temperature of the freeze-dryer to −20° C.Next, shelf temperature was increased by 10° C. each hour. The maximalshelf temperature is 20° C. The F&V mixture was completely dried in 4days. The dried mixture was pulverized with the same blender with thesame setting to obtain a fine powder and was stored in plastic bags at−80° C. freezer until it was use for animal diet preparation.

F&V powder was incorporated into experimental diets on a w/w basis,replacing 0, 5, 10 or 15% of the diet. Nutritional information about thediets are shown in Tables 2-5 as follows: calculated polyphenols contentin F&V powder (Table 2), nutrient content of animal diet with or withoutF&V powder (Table 3), polyphenol and total antioxidant content of animaldiet with or without F&V powder (Table 4), and fiber content includingtotal fiber and soluble and insoluble fiber of animal diet with orwithout F&V powder (Table 5).

Male C57BL/6J mice were assigned into one of 9 diet groups (12mice/group). Diets were fed ad libitum for 20 weeks; mice were weighedweekly and food intake was recorded.

At 15 weeks, total body fat and lean mass were measured by MM.

Mouse fecal sample was collected at 18 weeks and stored in −80° C.freezer for measurement of energy content by bomb calorimeter andmicrobiota analysis.

After 20 weeks, mice were euthanized. Blood sample was collected. Serumwere isolated and stored in −80° C. for further analysis. Liver andadipose tissue were partially fixed in formalin and partially firstfrozen in liquid nitrogen and then transferred to −80° C. for storage.Fixed liver and adipose tissue were processed for histopathology tomeasure lipid accumulation & inflammation, respectively.

Frozen adipose tissue samples were used to analyze mRNA levels ofpro-inflammatory molecules by RTqPCR.

Body Composition Analysis

Body composition (% fat tissue and % lean tissue) was assessed by usingrodent magnetic resonance imaging system (Whole Body CompositionAnalyzer, EchoMRI, Houston, Tex.) at 15 week of age.

Fecal Energy Density Assay

Fecal energy density was determined using a PARR 6200 Isoperibolcalorimeter (Parr Instrument Company, Moline, Ill., USA) following themanufacturer's instruction.

Adipose Tissue Histology and Adipocyte Size Determination

Epididymal adipose tissue were dissected, fixed, embedded in paraffin,and sectioned. The sections were stained with hematoxylin and eosin(H&E). Adipocyte size of H&E stained epididymal adipose tissue sectionswere measured based on previously reported method (Parlee et al., 2014).Briefly, the digital images of H&E-stained epididymal adipose tissueswere acquired with an Olympus FSX100 light microscope and the area (μm²)of each adipocyte were manually determined with touch-screen laptopcomputer. Data were expressed as the frequency of adipocytes compared tothe total number of adipocytes counted (% total).

Liver Tissue Histology and Hepatic Steatosis Area Measurement

Liver tissue were dissected, fixed, embedded in paraffin, and sectioned.The sections were stained with hematoxylin and eosin (H&E), which wasperformed in Animal Histology and Pathology Services at Tufts Universityand Tufts Medical Center for histology analysis. The digital images ofH&E-stained liver tissues were acquired with an Olympus FSX100 lightmicroscope. Hepatic steatosis area was measured using ImageJ software asprevious reported (McLaughlin et al., 2010).

Serum, Liver and Adipose Tissue Lipidomic Profile

Serum, liver and adipose tissue lipidomic profile was analyzed usingLC-MS/MS techniques by VCU Massey Cancer Center Lipidomics SharedResource.

Liver nSMase Activity Assay

Liver homogenates were prepared and liver nSMase activity was measuredbased on reported methods (Empinado et al., 2014). Briefly, nSMaseactivities were measured in liver homogenates, and 40 μg were used ineach assay. The protein concentration was assessed using a ThermoScientific Pierce BCA Protein Assay kit following the microplateprocedure. The nSMase activity assay was done in a 50 mM Tris-HCl (pH7.4) reaction buffer containing 7.5 mM MgCl₂, 10 μM C6-NBD-SM, 1 mMsodium ortovanadate, 15 mM sodium fluoride, protease inhibitor cocktailand 40 μg of homogenate in a final volume of 40 μl for 30 min. Thereactions were stopped by the addition of 0.5 ml methanol. After furtherincubation at 37° C. for 30 min, the samples were centrifuged at1,000×g, and the clear supernatant was transferred to clear HPLC vials.The generated fluorescent product, NBD-ceramide, was monitored by areverse phase HPLC.

Gene-Expression Analyses

Total RNA was extracted from liver and adipose tissue using TRIzolreagent (Invitrogen). Complementary DNA (cDNA) was generated byreverse-transcription of 1 μg total RNA using Super Script IIIFirst-Strand Synthesis System (Invitrogen). Gene expression levels ofinterest were quantitated by using SYBR Green reagent. Results arerepresented as a fold change in comparative expression level.

Measurement of Serum Pro-Inflammatory Cytokine Levels and Serum LipidsProfiling

Mouse serum pro-inflammatory cytokine levels were determined usingelectrochemiluminescent multiplex assays and serum lipids profiling wasperformed by Nutritional Evaluation Laboratory at HNRCA.

16S rDNA Microbiota ProfilingDNA Extraction and 16S rDNA Amplicon Generation

Bacterial genomic DNA was extracted using the QIAamp Stool DNA Kit(Qiagen, Germantown, Md.) with the following modifications. Samples wereresuspended in ASL buffer (Qiagen kit) in the presence of 500 mg of 0.1mm silica/zirconium beads (BioSpec Products, Bartlesville, Okla.) andincubated at 95° C. for 10 min. After cooling to room temperature, thesamples were placed on a bead-beater at 4° C. for 5 minutes. The stoolsolids were pelleted on a microfuge and the supernatant was treated withan Inhibitex tablet, after which the standard Qiagen protocol wasfollowed. Amplicons of the V4 region of the bacterial 16S ribosomal DNAwere generated by PCR, and amplicon pools were sequenced on a MiSeqsequencer (Illumina), and QIIME analysis were performed and an OTU tablewas generated by the Tufts University Core Facility Genomics Core.Shannon and Simpson diversity index were determined, and unweightedUniFrac analysis was conducted. Data were analyzed using BioconductorWorkflow. Kruskal-Wallis test was performed for each diversity metric,followed by a Wilcoxon Rank Sum test for pairwise comparisons with falsediscovery rate (FDR) correction (Callahan et al., 2016; Goodrich et al.,2014; Lozupone and Knight, 2008).

Microbiota Data Analysis

For analyzing microbiome data, a pipeline called Qiime (specificallyQIIME 1.8.0) was used. The basics of this pipeline are as follows: Allthe demultiplexed fastq files are combined into one file which includesjoining the paired end reads and concatenating all the files together(qiime.org/scripts/join_paired_ends.html) Barcodes are extracted fromeach read. (qiime.org/scripts/extract barcodes.html) The libraries aresplit which generates a file where each sequence is identified with acorresponding identifier from the mapping file.(qiime.org/scripts/split_libraries.html) From there, OTU picking isperformed using a closed reference, which generates the OTU table. Italso gives a table with the sequence counts per sample which is usedwhen deciding the sampling depth in the alpha analysis.(qiime.org/scripts/pick_closed_reference_otus.html). The reference OTUscome from a database called greengenes and the most recent release whichis greengenes_13_8 (greengenes.secondgenome.com/downloads) was used.Also, a 0.99 similarity was used when picking the OTUs.

Quantification and Statistical Analysis

Data are presented as mean±SE. A two-tailed unpaired t-test ortwo-tailed unpaired t-test with Welch's correction was used forcomparison between B0 and B15 with equal or unequal variance,respectively. The differences among MC, HF0 and HF15 data were analyzedby one way ANOVA followed by Dunnett's post-hoc test. Correlationcoefficients were calculated by using a nonparametric Spearman's rankcorrelation; and p values from Spearman correlation analysis of gutbacterial abundance and clinical biomarkers were corrected for falsedetection rate using the Benjamin-Hochberg method. Differentialabundance of gut bacteria between groups was analyzed using Deseq2package. Significance was set at p<0.05.

TABLE 1 Correlations between sphingolipids and TNFα and hepaticsteatosis area Basal diet High fat diet Factors R value P value R valueP value Hepatic steatosis area × Total serum ceramides 0.755 0.005 0.7620.004 Hepatic steatosis area × TNF-α 0.762 0.004 0.529 0.024 Hepaticsteatosis area × serum C16:0 ceramide 0.473 0.142 0.657 0.020 TNF-α ×serum C16:0 ceramide 0.254 0.242 0.409 0.047 Hepatic steatosis area ×serum C20:0 ceramide 0.669 0.035 0.531 0.075 Hepatic steatosis area ×serum C22:0 ceramide 0.748 0.005 0.392 0.208 Hepatic steatosis area ×serum C24:0 ceramide 0.664 0.018 0.469 0.124 Hepatic steatosis area ×serum C24:1 ceramide 0.399 0.199 0.720 0.008 TNF-α × serum C24:1ceramide 0.239 0.261 0.516 0.010

Correlation Analysis was Conducted Using Spearman's Correlation Test

TABLE 2 Calculated polyphenols content in F & V powder F &V polyphenolcontent μg/g % Flavanones 463.0  25.64% Hydroxycinnamic 419.5  23.23%acids Anthocyanins 322.8  17.88% Flavonols 230.2  12.75% Isoflavonoids184.8  10.23% Flavanols 118.5   6.56% Lignans 42.8   2.37%Hydroxybenzoic 13.8   0.77% acids Flavones 7.7  0.43% Stilbenes 1.5 0.08% Other 1.2  0.07% Total 1805.7   100.0%The polyphenols content in F&V powder is calculated based on USDAdatabase at www.ars.usda.gov/nutrientdata

TABLE 3 Nutrient content of animal diet with or without F & V powder F &V Energy Diet Content Protein Carb Fat Density Group* (%, w/w) (kcal %)(kcal %) (kcal %) (kcal/g) B0   0 20.3 64   15.8 4.00 B5   5 19.9 64.815.3 3.97 B10 10 19.4 65.7 14.9 3.94 B15 15 19   66.5 14.5 3.91 MC  020.0 70.0 10.0 3.85 HF0   0 20.0 35.0 45.0 4.73 HF5   5 19.6 37.3 43.14.66 HF10 10 19.2 39.6 41.2 4.59 HF15 15 18.7 41.9 39.3 4.53 *B0 (basaldiet); B5 (basal diet + 5% F & V); B10 (basal diet + 10% F & V); B15(basal diet + 15% F & V); MC (low fat diet); HF0 (high fat diet + 0% F &V); HF5 (high fat diet + 5% F & V); HF10 (high fat diet + 10% F & V);HF15 (high fat diet + 15% F & V)

TABLE 4 Polyphenol and total antioxidant content of animal diet with orwithout F & V powder ¹ Total Total Total antioxidant antioxidant Dietpolyphenols ² nutrients ³ nutrients ³ Group* (μg/g) (μg/g) (μg/kcal) B0  0 105.2  26.3  B5  114 196.1  53.4  B10 227 287.1  80.4  B15 341 378.0 107.5  MC  0 75.2  19.6  HF0   0 92.4  19.6  HF5  114 184.0  46.9  HF10227 275.5  74.3  HF15 341 367.1  101.7  ¹ The F & V powder containstotal polyphenols 2271 μg/g and total antioxidant nutrients 1923.5 μg/g.² The F & V powder contains 121 polyphenolic compounds. The top 10, i.e.hesperetin, caffeoylquinic acid, quercetin, malvidin, genistin, daidzin,naringenin, pelargonidin, cyanidin, and lariciresinol, constitute 84.9%of total polyphenols. ³ Total antioxidant nutrients include vitamin C,E, Se, and Zinc. *B0 (basal diet); B5 (basal diet + 5% F & V); B10(basal diet + 10% F & V); B15 (basal diet + 15% F & V); MC (low fatdiet); HF0 (high fat diet + 0% F & V); HF5 (high fat diet + 5% F & V);HF10 (high fat diet + 10% F & V); HF15 (high fat diet + 15% F & V) Thecontent of polyphenols and antioxidant nutrients in F & V powder iscalculated based on USDA database athttp://www.ars.usda.gov/nutrientdata

TABLE 5 Fiber content of animal diet with or without F&V powder TotalTotal Soluble Soluble Insoluble Insoluble Diet fiber fiber fiber fiberfiber fiber Group* (μg/g) (μg/kcal) (μg/g) (μg/kcal) (μg/g) (μg/kcal) B050.0 12.5 0.0 0.0 50.0 12.5 B5 49.0 12.3 0.3 0.1 48.7 12.2 B10 48.0 12.10.7 0.2 47.4 11.9 B15 47.1 12.0 1.0 0.3 46.1 11.7 MC 47.4 12.3 0.0 0.047.4 12.3 HF0 58.3 12.3 0.0 0.0 58.3 12.3 HF5 56.9 12.2 0.3 0.1 56.512.1 HF10 55.5 12.0 0.7 0.2 54.8 11.8 HF15 54.1 11.8 1.0 0.3 53.1 11.5F&V 30.2 8.9 6.5 1.9 23.7 7.0 *B0 (basal diet); B5 (basal diet + 5%F&V); B10 (basal diet + 10% F&V); B15 (basal diet + 15% F&V); MC (lowfat diet); HF0 (high fat diet + 0% F&V); HF5 (high fat diet + 5% F&V);HF10 (high fat diet + 10% F&V); HF15 (high fat diet + 15% F&V)

Results F&V Supplementation Reduced Weight Gain in Obese Mice Fed the HFDiet, but had No Effect on Weight Gain in Lean Mice Fed the Basal Diet

To investigate the effects of F&V on obesity and metabolic diseases, aunique F&V mixture containing 24 of the most commonly consumed F&V basedon USDA census data (average per capita daily consumption (grams) from1994 to 2008) was formulated. To mimic the natural patterns ofconsumption, the proportion of each individual fruit or vegetable in theF&V mixture was calculated based on the relative proportion of yearlyF&V consumption in the United States. A blend of 12 fruits and 12vegetables was freeze-dried, pulverized, and incorporated intoexperimental diets on a w/w basis, replacing 0, 5, 10 or 15% of thediet.

Next, the effects of the F&V supplementation in lean mice fed standarddiet (Basal, B; AIN-93, 16 kcal % fat) or obese mice was calculated.Obesity was induced by feeding HF diet (45 kcal % fat, which istypically used in studies to induce obesity in mice). Because there areslight differences between the AIN-93 and the HF diet used, anadditional group of mice were fed a micronutrient matched control diet(MC; 10 kcal % fat), as a control for the high fat diet (Warden andFisler, 2008). Mice were fed their respective diets for 20 weeks.

Obese mice fed HF diet alone (HF0) gained 77.1% more weight over the 20weeks than those fed the MC diet; however, mice fed the HF dietsupplemented with 15% F&V (HF15) gained significantly less body weightcompared to mice fed HF diet without F&V (HF0) (FIG. 1B). No significantdifferences were observed on weight gain in mice fed the HF diet withthe lower (5% and 10%) levels of F&V supplementation (FIG. 1B),indicating that 15% F&V is the most efficacious amount in reducing bodyweight gain in diet-induced obesity. Similar observation was made onother outcomes of interest. Accordingly, data from 15% supplementeddiets is used. Further, body composition (measured using Mill at 17weeks of age) showed that, compared to mice fed the MC diet, those fedthe HF diet had 107% more fat tissue weight (FIG. 1D) and 58.3% moretotal fat mass as a percentage of body weight (FIG. 1F). The percentageof lean tissue mass in mice fed the HF diet (HF0) was 18.5% lower thanthat of the mice fed the MC diet (FIG. 1H). Mice fed the 15% F&Vsupplemented HF diet had significantly less fat tissue weight (FIG. 1D)compared to those fed the HF diet alone (HF0).

Mice fed the basal diet alone (B0) had lower weight gain than those fedHF0 diet (FIGS. 1A&B). There was no significant effect of F&Vsupplementation on weight gain (FIG. 1A) and body composition (FIGS. 1C,1E, 1G, and 1I) in mice fed the Basal diet.

There was no significant impact of F&V on the level of food energyintake (Figure S1D) in obese mice. F&V supplementation in both basal andHF diets significantly increased fecal weight (Figure S1E and S1F),fecal energy density (Figure S1G and S1H), and fecal energy excretion(Figure S1I and S1J), indicating that beneficial effects of F&V onreducing weight gain in mice fed HF diet may be mediated throughdecreasing energy harvesting.

Dietary F&V Supplementation Reduced Adipose Tissue Inflammation andPrevented NAFLD Independent of Food Intake and Body Weight Reduction

Dysfunctional adipose tissue, especially visceral white adipose tissue,characterized by adipocyte death and infiltrated macrophages and otherinflammatory cells, is linked to pathogenesis of metabolic diseases(Paniagua, 2016; van Greevenbroek et al., 2016). Crown-like structures,known to be formed by accumulated inflammatory immune cells around dyingadipocytes, in gonadal adipose tissue, have been associated with thedevelopment of metabolic disorders (van Beek et al., 2015). Immune cellfiltration is a prominent feature of adipose tissue dysfunction (Guziket al., 2017).

To explore whether increasing F&V consumption prevents adipose tissueinflammation, histological analysis of epididymal adipose tissue (eAT)in mice fed Basal or HF diets supplemented with or without F&V wasperformed. Crown like structures were found in mice fed either the HF0diet or B0 diets, albeit at much lower densities in the B0 compared toHF0 (FIGS. 2A and 2D). Feeding mice the HF or basal diets supplementedwith 15% F&V eliminated the crown-like structures (FIGS. 2B and 2E).These data indicate that the anti-inflammatory effect of F&V in eAT isindependent of its anti-obesity effect due to the fact that there was nodifference in average body weight between mice fed a basal diet withoutand with 15% F&V (FIGS. 1A and 1B). Similar to previous reports(Strissel et al., 2007), a prevalence of small adipocytes (<2000 μm²)was observed in mice fed a HF diet at 20 weeks (FIG. 2F). Increasedprevalence of small adipocytes is a feature of dead adipocytes inremodeled eAT and is often associated with NAFLD due to reduced fatstorage capacity of the adipose tissue. Adding 15% F&V to HF dietremarkably reduced the prevalence of small adipocytes induced by HF diet(FIG. 2F). There was no significant effect of F&V on the eAT adipocytesize in the mice fed the Basal diet (FIG. 2C).

Pro-inflammatory cytokine gene expression levels were examined in themice eAT and it was found that the mRNA levels of pro-inflammatorycytokines, TNFα, IL-1β, IL-6, and MCP-1, were higher in eAT of the micefed HF diet alone compared to those fed MC diet. Compared to HFD alone,mice fed HFD with 15% F&V had significantly less pro-inflammatorycytokine mRNA expression in eAT (Figure S2A-S2D). No significantdifference in the pro-inflammatory cytokine mRNA levels between mice fedB0 and B15 diets was observed.

H&E histological staining of liver sections showed no sign of NAFLD inmice fed the MC diet (10 kcal % fat) (FIG. 3A). Mice fed either theBasal diet (B0, AIN-93 with 16 kcal % fat) or HF diet (HF0, 45 kcal %fat) for 20 weeks developed mild and severe NAFLD, respectively (FIGS.2H and 2K). In comparison, mice receiving 15% F&V in both the basal andHF diets showed significantly less hepatic steatosis (p<0.05, FIGS. 2I,2J, 2L, and 2M). These data indicate that the anti-hepatosteatosiseffect of F&V is independent of its anti-obesity effect due to the factthat there was no difference in average body weight between mice fed abasal diet without and with 15% F&V (FIGS. 1A and 1B).

As dyslipidemia is strongly associated with NAFLD (Chatrath et al.,2012; Katsiki et al., 2016; Zhang and Lu, 2015), serum lipid profilingwas performed. Results show that F&V supplementation had no significanteffect on serum lipids profiles (FIG. 13A-13J). Thus, the effect of F&Vin this model on liver steatosis is not mediated through changes inlipid profile.

Compared to mice fed HFD alone, mice fed HFD+F&V had lower spleen weightand spleen weight index, which was calculated as mg spleen/g body weight(FIG. 8).

F&V Supplementation Reduced Circulating and Liver Tissue Ceramides asWell as Pro-Inflammatory Cytokine TNFα

As levels of circulating sphingolipids, especially ceramides, areclosely associated with NAFLD pathogenesis (Ilan, 2016;Nikolova-Karakashian, 2018; Regnier et al., 2018), serum sphingolipidprofile analysis was performed. The results show that the mice fed theB15 (FIG. 3A) or HF15 diets (FIG. 3B) had significantly lower serumlevels of total ceramides compared to B0 or HF0, respectively.

Similarly, serum levels of other ceramide species, such as C16:0ceramide, C24:0 ceramide, and C24:1 ceramide, in the mice fed B15 orHF15 diets were significantly lower or trended to be lower compared toB0 or HF0, respectively (FIG. 3E-3F, 3Q-3R, 3U-3V). No significantdifferences of the levels of serum C18:1 ceramide and C18:1 ceramidewere found in the mice fed B0 diet compared to that of B15 diet or themice fed HF diet compared to that of HF15 diet (FIGS. 3I-3J and 3M-3N).Spearman correlation analysis indicated that NAFLD in mice fed eitherthe Basal or HF diets is positively correlated with total serumceramides and TNFα, respectively (Table 1). Serum long chain ceramidespecies including C16:0, C20:0, C22:0, C24:0, and C24:1 ceramides arealso positively correlated with NAFLD in mice fed either the basal or HFdiets (Table 1). These results indicate that the effects of F&V on NAFLDmay be mediated through changes in ceramide levels.

Levels of liver ceramides were measured. It was found that mice fed theHF0 had higher levels of liver total ceramide and other ceramide speciescompared to mice fed the MC diet. HF diet-induced higher ceramide levelswere attenuated by adding 15% F&V to the HF diet (FIG. 3D, 3H, 3L, 3P,3T, 3X). No significant impact of F&V on liver ceramide levels in micefed the Basal diet (FIG. 3C, 3G, 3K, 3O, 3S, 3W) was observed, indicatedthat there are differences between impact of F&V on systemic and tissuelevels of ceramides.

To determine the mechanism of F&V-induced reduction in liver ceramides,its effects on mRNA levels of liver ceramide synthase (CerS), the enzymerequired for ceramide generation from either de novo synthesis pathwayor salvage pathway were assayed. CerS mRNA levels of the mice fed HF0and HF15 diet were assayed, since more dramatic effect of F&V wasobserved in mice fed the high fat diet. No differences in liver CerS2,CerS5, and CerS6 mRNA levels were found between mice fed the HF dietalone and mice fed the HF diet supplemented with 15% F&V (FIG. 14A-14C),indicating that the effect of F&V in reducing liver ceramide levels wasnot due to altered transcripts levels of CerS genes. While these resultscannot rule out the possibility that F&V might alter CerS enzyme proteinlevels and/or activity, it is contemplated that the effect of F&V onceramide level is due to reduction in activity of the enzymesphingomyelinase (SMase), the enzyme that catalyzes the release ofceramide from sphingomyelin. Support for the hypothesis comes from thefact that F&V are rich in antioxidants, and SMase is regulated by redoxhomeostasis (Cinq-Frais et al., 2013; Martin et al., 2007). The activityof the liver neutral SMase (nSMase) was assayed and it was found thatmice fed the HF0 diet alone had higher liver nSMase activity compared tomice fed the MC diet, and that mice fed the HF diet supplemented with15% F&V had significantly lower nSMase activity compared to HF0 (FIG.4E). No effect of increasing F&V consumption on liver nSMase activitywas observed in mice fed the basal diet (FIG. 4D). These results areconsistent with the results on the impact of obesity and F&V on liverceramide levels (FIG. 3) and support that obesity induced increase inceramide level and its reduction by F&V is mediated through decrease innSMase activity.

Activation of liver FXR, a key regulator controlling various livermetabolic processes, suppresses liver inflammation by inhibiting NF-κBtarget inflammatory genes including TNFα (Y D et al., 2008). On theother hand, TNFα and HFD feeding down-regulate liver FXR expression(Geier et al., 2005; Kim et al., 2003; Nie et al., 2017). FXR agonistcould upregulate HFD-induced down-regulation of FXR expression andinhibit TNFα and NF-κB signaling pathway (Hu et al., 2018). TNFα hasbeen shown to stimulate a neutral plasma membrane-associated SMaseactivity leading to ceramides generation (Schutze et al., 1994) andcontribute to the development and progression of NAFLD (De Taeye et al.,2007; Kakino et al., 2018). Furthermore, TNFα and ceramides are shown toplay critical roles in the development of metabolic disorders includingNAFLD and diabetes (Ilan, 2016; Rehman et al., 2017; Schmidt-Arras andRose-John, 2016). Inflammatory cytokines TNFα and ceramide are alsoengaged in regulating each other's level. Further, ceramide can increasemitochondrial generation of reactive oxygen species resulting ininflammation and metabolic disorder such as NAFLD (Pagadala et al.,2012). Thus, to have a better understanding of underlying mechanism ofobesity and F&V induced changes in adipose tissue inflammation andNAFLD, the levels of circulating and liver TNFα and liver FXR mRNAlevels of mice fed a basal or HF diet supplemented with or without 15%F&V as well as mice fed MC diet were determined. It was found that,compared to the mice fed MC diet, those fed the HF diet alone showedsignificantly higher circulating and liver TNFα protein levels, lowerliver FXR mRNA, and higher liver nSMase specific activity (FIG. 4A-4D).Feeding mice HF diet with 15% F&V prevented HF diet-induced decrease inliver FXR mRNA expression (FIG. 4B) as well as increase in liver nSMasespecific activity (FIG. 4D) and trended to lower serum and liver TNFαlevels (P=0.0761 and P=0.0971, respectively; FIGS. 4A and 4C). The micefed the Basal diet with 15% F&V had significantly lower serum TNFαlevels compared to those fed the Basal diet alone (p<0.001, FIG. 4E),but there were no dramatic differences in liver FXR mRNA levels, liverTNFα protein levels, and liver nSMase specific activity between mice fedthe B0 1 diet and mice fed the B15 diet (FIG. 4F-4G). These resultsindicate that the preventive effect of F&V supplementation on NAFLDmaybe partially mediated by modulating anti-inflammation gene theexpression of the anti-inflammation gene, FXR, TNFα, and ceramideslevels. This is supported by the result of the correlation analysisdiscussed below.

Compared to mice fed HFD alone, mice fed HFD supplemented with F&V hadsignificantly higher serum levels of LXA4 and 14,15-EET (by 203% and96%, respectively) and lower serum levels of 20-HETE and DHGLA (by 14%and 41%, respectively) (FIG. 9).

Spearman correlation analysis indicated significant positivecorrelations between TNFα levels and total ceramides, as well as C16:0,C20:0, C22:0, C24:0, and C24:1 ceramide species (Table 1). Thisindicates that TNFα may be involved in both HF diet-, and F&V-inducedchanges in ceramide levels. A significant positive correlation was alsoobserved between TNFα and NAFLD (Table 1), supporting a link betweenTNFα, ceramide and NAFLD.

Gut Microbiota Dysbiosis was Mitigated by F&V Supplementation and wasAssociated with Biochemical and Clinical Outcomes

To evaluate the effects of F&V supplementation on the gut microbiota, weperformed 16S rRNA gene-based taxonomic profiling. Compared to mice fedBasal or HF diets alone, we found that mice fed the Basal or HF dietssupplemented with F&V had significantly higher alpha diversity in fecalmicrobiota (FIGS. 5A and 5C). Principal Coordinate Analysis showedshifts in gut microbiota composition according to diet, with fecalmicrobiota composition differing the most according to F&V consumptionin both diets (FIGS. 5B and 5D).

Furthermore, we observed changes in gut bacteria composition at alltaxonomic levels.

Compared to mice fed the MC diet, those fed the HF diet had dramaticallydifferent gut bacteria composition. While mice fed the F&V-supplementedHF diet had microbial composition more similar to that of mice fed theMC diets than those fed the HF diet. Microbial composition of mice fedthe F&V-supplemented basal diet had a different pattern from that ofmice fed Basal diet (FIG. 6A-6E).

Differential abundance analyses revealed a significantly higherabundance of following taxonomic groups in mice fed the HF15 dietcompared to those fed the HF0 diet: Anaeroplasma (10×), Leuconostoc(7.5×), Trichococcus (5×), Oscillospira (˜1.5×), 2 groups annotated tothe Firmicutes phylum (˜2×), one to the Bacteroidetes (2.5×) phylum, andone Cyanobacteria (7.5×). In contrast, several genera had lowerabundances in mice fed the HF15 diet compared to those fed the HF0 diet:Lactococcus (˜2.5×), SMB53 (−8×), and one unannotated group from theFirmicutes phylum (−7×), and one from the Bacteroidetes phylum (−4×)(FIG. 15A). Anaeroplasma, Leuconostoc, Trichococcus, and the unannotatedgenus from the Cyanobacteria phylum were also more abundant in theHF15-diet fed mice compared to the MC-diet fed mice (10×, 7.5×, 6×, and7× more abundant, respectively, FIG. 15B). Similarly, the Anaeroplasmaand Leuconostoc genera were more abundant in the B15 diet-fed micecompared to the B0 diet-fed mice (FIG. 15C).

Spearman correlation analyses showed that gut bacteria, which werepositively correlated with F&V intake in mice fed the Basal diets (B0 &B15) were inversely associated with inflammation, ceramides, andpositively associated with fecal energy excretion (FIG. 7A). Similarly,in mice fed the HF diet, bacteria that were positively correlated withF&V intake were positively correlated with a healthier metabolicprofile, as seen by liver FXR mRNA expression and percent lean bodymass, and negatively associated with fat mass, inflammation, andceramides (FIG. 7B).

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Example 2 Dietary Fruit and Vegetable Supplementation SuppressDiet-induced Atherosclerosis in LDL Receptor Knockout Mice Materials andMethods Fruits and Vegetables (F&V) Powder Preparation

F&V mixture containing a combination of 24 of the most commonly consumedF&V based on USDA census data was homogenized to prepare thefreeze-dried powder as described in Example 1.

Animals and Diets

Four-week-old male LDL receptor knockout mice (B6.129S7-Ldlr^(tm1Her)/J,stock number 002207) were purchased from The Jackson Laboratory (BarHarbor, Me., USA) and housed at the animal care facility at Jean MayerUSDA Human Nutrition Research Center on Aging at Tufts University. After12 days of acclimation, individually caged animals were assigned intoweight-matched three groups, including low fat diet (LF) group, high fatdiet (HF0) group, and high fat diet supplemented with 15% of F&V mixture(HF15) group. The LF diet was made of modified AIN-93M diet, with 10%kcal from fat (4.4% kcal from cocoa butter/5.6% kcal from soybean oil,Research Diets, #D17030910M) and 52 mg cholesterol/1000 kcal added. TheHF0 diet was consisted of modified AIN-93M diet, with 27% kcal from fat(24.2% kcal from cocoa butter/2.8% kcal from soybean oil, ResearchDiets, # D17030911M) and 130 mg cholesterol/1000 kcal included. The HF15diet was prepared by replacing 15% HF0 diet with 15% F&V mixture (w/w)(equivalent to 8-9 servings of F&V/day for humans). Mice were fed adlibitum with respective diets for 20-weeks. The body weight wererecorded weekly, and fecal sample was collected for gut microbiomeanalysis.

Animal Blood Collection and Organ Isolation

After 20 weeks, mice were euthanized. Blood sample was collected bycardiac puncture from each animal, and serum were isolated and stored in−80° C. for further analysis. Subsequently, the thoracic cavity wasopened, and the aorta was isolated as previously reported [1]. Liver wasdissected out and weighed, then sectioned into two pieces, one informalin, and one in foil then frozen in liquid nitrogen and thentransferred to −80° C. for storage.

Aortic Lesion Area Quantification

The isolated descending aorta, stored in a 10% buffered formaldehydesolution, were cleared of fat and cut longitudinally, and then pinneddown on a black wax platform using insect pins. Aortic lesions werevisualized through staining with freshly prepared Oil Red O inisopropanol. Resulting images were captured under a dissectionmicroscope. Aortic atheroma lesion area was evaluated with AdobePhotoshop CC 2015 software (Adobe Systems, Mountain View, Calif.), andthe ratios of the plaque area stained with oil red O over total aortaarea were quantitated.

Hepatic Steatosis Analysis

Fixed liver tissue were processed for histopathology to measure lipidaccumulation. Hepatic steatosis area was quantitated with formalin-fixedH&E stained sections, and the percentage of the lipid area to the totalarea was calculated using ImageJ (Neuberger and James 1999).Non-hepatocyte areas such as sinusoids, portal tracts, and hepatic veinswere excluded for the analysis.

Gene-Expression Analyses

Total RNA was extracted from frozen liver tissue using TRIzol reagent(Invitrogen). Complementary DNA (cDNA) was generated byreverse-transcription of 1 μg total RNA using Super Script IIIFirst-Strand Synthesis System (Invitrogen). Gene expression levels ofinterest were quantitated by using SYBR Green reagent. Results arerepresented as a fold change in comparative expression level. Sequencesof forward or reverse oligonucleotide primers are listed below in Table6.

TABLE 6 Official Symbol Gene ID Forward Sequence Reverse Sequence Hprt1Hprt 15452 AAGCTTGCTGGTGAAAAGGA TTGCGCTCATCTTAGGCTTT (SEQ ID NO: 1)(SEQ ID NO: 2) TNFα Tot 21926 TTGCTCTGTGAAGGGAATGGGGCTCTGAGGAGTAGACAATAAAG (SEQ ID NO: 3) (SEQ ID NO: 4) FAS Fasn 14104CCCTTGATGAAGAGGGATCA ACTCCACAGGTGGGAACAAG (SEQ ID NO: 5) (SEQ ID NO: 6)

Measurement of Serum Pro-Inflammatory Cytokine Levels and Serum LipidsProfiling

Mouse serum pro-inflammatory cytokine levels were determined usingelectrochemiluminescent multiplex assays and serum lipids profiling wasperformed by Nutritional Evaluation Laboratory at HNRCA.

16S rDNA Microbiota Profiling

Bacterial genomic DNA was extracted using the QIAamp Stool DNA Kit(Qiagen, Germantown, Md.) following manufacturer's instruction.Amplicons of the V4 region of the bacterial 16S ribosomal DNA weregenerated by PCR, and amplicon pools were sequenced on a MiSeq sequencer(Illumina). QIIME analysis were performed and an OTU table was generatedby the Tufts University Core Facility Genomics Core. Shannon and Simpsondiversity index were determined, and unweighted UniFrac analysis wasconducted. Data were analyzed using Bioconductor Workflow.Kruskal-Wallis test was performed for each diversity metric, followed bya Wilcoxon Rank Sum test for pairwise comparisons with false discoveryrate (FDR) correction [2-4].

Statistical Analysis

Data are presented as mean±SE and were analyzed by one-way ANOVAfollowed by Dunnett's post-hoc test. Correlation coefficients werecalculated by using a nonparametric Spearman's rank correlation; and pvalues from Spearman correlation analysis of gut bacterial abundance andclinical biomarkers were corrected for false detection rate using theBenjamini-Hochberg method. Differential abundance of gut bacteriabetween groups was analyzed using Deseq2 package. Significance was setat p<0.05.

Results F&V Supplementation Suppressed High Fat Diet-Induced AorticAtherosclerosis in LDLR KO Mouse.

To determine the effectiveness of F&V supplementation on atheroscleroticlesion formation in LDLR KO mice, aortic atherosclerosis lesion area wasmeasured by en face Oil Red O staining. Mice fed HF0 diet had largeraortic atherosclerotic lesion area than mice fed LF diet (6.5 foldincrease). Compared to mice fed HF0, the aortic lesion steatosis in micefed HF15 diet reduced more than 80% (FIG. 17). These results indicatethat F&V supplementation suppressed high fat diet-induced aorticatherosclerosis in LDLR KO mouse.

F&V Supplementation Prevented High Fat Diet-Induced Hepatic Steatosis inLDLR KO Mouse

There were no significant differences with body weight among threegroups (FIG. 18A). However, liver weight and the ratio of liver weightover final body weight were higher in mice fed HF0 diet than in mice fedLF diet. Mice fed HF15 diet had lower liver weight and the ratio ofliver weight over final body weight compared to mice fed HF0 diet (FIGS.18B and 18C). The effects of FV on hepatic steatosis were examined. Micefed HF0 diet had larger hepatic steatosis area than mice fed LF diet(1.9 fold increase). Compared to mice fed HF0 diet, the hepaticsteatosis area in mice fed HF15 diet reduced more than 80% (FIG. 19).

Effects of F&V Supplementation on Suppression of Aortic Atherosclerosisand Prevention of Hepatic Steatosis are Associated with Improvement ofDiet-Induced Dyslipidemia and Reduction of Serum TNFα Levels in LDLR KOMouse

As dysregulated lipids metabolism is associated with pathogenesis ofatherosclerosis, plasma lipid profile was assessed. Mice fed HF0 diethad significantly higher plasma triglyceride (TG) and low-densitylipoprotein (LDL) cholesterol and lower high-density lipoprotein (HDL)cholesterol levels than mice fed LF diet. Mice fed HF15 dietsignificantly improved dyslipidemia to the levels similar to LF-fed mice(FIG. 20A-4E). Furthermore, the ratio of TG/HDL (FIG. 20F), LDL/HDL(FIG. 20G), and non HDL/HDL (FIG. 20H) were higher in mice fed HF0 thanin mice fed LF. Mice fed HF15 diet had lower ratio of TG/HDL (FIG. 20F),LDL/HDL (FIG. 20G), and non HDL/HDL (FIG. 20H) than mice fed HF0 diet,indicating that F&V improved diet-induced dyslipidemia in LDLR KO mouse.

It was investigated whether effects of F&V supplementation onsuppression of high fat diet-induced aortic atherosclerosis andprevention of hepatic steatosis in LDLR KO mouse is mediated throughreduction of circulating pro-inflammatory cytokine levels. F&Vsupplementation significantly reduced serum TNFα levels (FIG. 21A).Other pro-inflammatory cytokine levels, such as serum IL-6 (FIG. 21B),IL-1β (FIG. 21C), and KC/GRO (FIG. 21D), trended to be lower in mousefed HF15 diet compared to those fed HF0 diet.

Since F&V supplementation alleviated dyslipidemia and reduced serum TNFαlevels, mRNA levels of liver fatty acid synthase (Fasn), a key lipogenicenzyme involved in de novo lipid biosynthesis, and TNFα, which playscritical role in fatty liver pathogenesis and may be involved inatherogenesis [5-8] were determined. It was found that mice fed HF0 diethad higher mRNA levels of Fasn and TNFα in liver tissue than mice fed LFdiet (FIGS. 22A and 22B). Compared to mice fed HF0 diet, mice fed HF15diet had significant lower expression levels of these two genes (FIGS.22A and 22B).

Circulating pro-inflammatory cytokine TNFα levels and dyslipidemia areknown to play critical roles in pathogenesis of atherosclerosis [5, 6,9-11] and hepatic steatosis [7, 12, 13]. Therefore, Spearman correlationanalysis was performed. It was found that aortic atherosclerotic lesionand hepatic steatosis area were negatively correlated with plasma HDL(p<0.001, respectively) and positively and significantly associated withTNFα and ratio of LDL/HDL, TG/HDL, and non HDL/HDL (FIG. 22). Ourresults indicate that the F&V mixture prevented HF-inducedatherosclerosis and hepatic steatosis, which may be mediated by reducingplasma TNFα levels and improving dyslipidemia.

F&V Supplementation Mitigated Gut Microbiota Dysbiosis in LDLR KO Mouse

To evaluate the effects of F&V supplementation on the gut microbiota,16S rRNA gene-based taxonomic profiling was performed. Compared to micefed HF diets alone, it was found that mice fed the HF diets supplementedwith F&V had significantly higher alpha diversity in fecal microbiota(FIG. 24A). Principal Coordinate Analysis showed shifts in gutmicrobiota composition of F&V consumption compared to both LF and HFdiets (FIG. 24B).

REFERENCES FOR EXAMPLE 2

-   1. Meydani, M., et al., Long-term vitamin E supplementation reduces    atherosclerosis and mortality in Ldlr−/− mice, but not when fed    Western style diet. Atherosclerosis, 2014. 233(1): p. 196-205.-   2. Callahan, B. J., et al., Bioconductor Workflow for Microbiome    Data Analysis: from raw reads to community analyses. F1000Res, 2016.    5: p. 1492.-   3. Goodrich, J. K., et al., Conducting a microbiome study.    Cell, 2014. 158(2): p. 250-262.-   4. Lozupone, C. A. and R. Knight, Species divergence and the    measurement of microbial diversity. FEMS Microbiol Rev, 2008.    32(4): p. 557-78.-   5. Ohta, H., et al., Disruption of tumor necrosis factor-alpha gene    diminishes the development of atherosclerosis in ApoE-deficient    mice. Atherosclerosis, 2005. 180(1): p. 11-7.-   6. Branen, L., et al., Inhibition of tumor necrosis factor-alpha    reduces atherosclerosis in apolipoprotein E knockout mice.    Arterioscler Thromb Vasc Biol, 2004. 24(11): p. 2137-42.-   7. Paredes-Turrubiarte, G., et al., Severity of non-alcoholic fatty    liver disease is associated with high systemic levels of tumor    necrosis factor alpha and low serum interleukin 10 in morbidly obese    patients. Clin Exp Med, 2016. 16(2): p. 193-202.-   8. Seo, Y. Y., et al., Tumor Necrosis Factor-alpha as a Predictor    for the Development of Nonalcoholic Fatty Liver Disease: A 4-Year    Follow-Up Study. Endocrinol Metab (Seoul), 2013. 28(1): p. 41-5.-   9. Zhang, Y., et al., TNF-alpha promotes early atherosclerosis by    increasing transcytosis of LDL across endothelial cells: crosstalk    between NF-kappaB and PPAR-gamma. J Mol Cell Cardiol, 2014. 72: p.    85-94.-   10. McKellar, G. E., et al., Role for TNF in atherosclerosis?    Lessons from autoimmune disease. Nat Rev Cardiol, 2009. 6(6): p.    410-7.-   11. Boesten, L. S., et al., Tumor necrosis factor-alpha promotes    atherosclerotic lesion progression in APOE*3-Leiden transgenic mice.    Cardiovasc Res, 2005. 66(1): p. 179-85.-   12. Diehl, A. M., Tumor necrosis factor and its potential role in    insulin resistance and nonalcoholic fatty liver disease. Clin Liver    Dis, 2004. 8(3): p. 619-38, x.-   13. Kakino, S., et al., Pivotal Role of TNF-alpha in the Development    and Progression of Nonalcoholic Fatty Liver Disease in a Murine    Model. Horm Metab Res, 2018. 50(1): p. 80-87.

Example 3

Experiments are conducted to determine the effect of the F&Vcompositions described herein on lifespan of mice fed basal and HFD. Itis contemplated that the compositions increase life and health span ofmice and that the impact is more dramatic in those fed obesogenic diet.Similar to obesity, in aging, there is increase in oxidative stress,inflammation, increase in ceramide and alteration of gut microbiota.Many of the chronic and infectious disease associated with aging arealso seen in obesity.

All publications, patents, patent applications and accession numbersmentioned in the above specification are herein incorporated byreference in their entirety. Although the invention has been describedin connection with specific embodiments, it should be understood thatthe invention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications and variations of thedescribed compositions and methods of the invention will be apparent tothose of ordinary skill in the art and are intended to be within thescope of the following claims.

1. A composition, comprising: a dry powder comprising a mixture of fruitspecies and a mixture of vegetables species, wherein the fruit speciesare six or more fruit species selected from oranges, apples, bananas,grapes, watermelon, pineapple, strawberries, cantaloupe, lemons,grapefruit, peaches, and pears; and wherein the vegetable species arethree six or more vegetable species selected from potatoes, tomatoes,sweet corn, onions, head lettuce, romaine, bell peppers, carrots,cucumbers, cabbage, beans, and sweet potato.
 2. The composition of claim1, wherein said mixture is a dry powder.
 3. The composition of claim 1,wherein the mixture comprises 40-65 grams of dry powder.
 4. Thecomposition of claim 1, wherein the mixture comprises 12 fruit speciesand 12 vegetable species.
 5. The composition of claim 1, wherein saidmixture of fruit and vegetable species comprises 16-20% oranges, 14-18%tomatoes, 8-11% apples, 13-16% potatoes, 3.0-5.5% bananas, 3-4% sweetcorn, 3-4% grapes, 2-3% lettuce, 1-2% escarole, 1-2% brussels sprouts,1-2% cabbage, 1-2% carrots, 1-3% onions, 1-2% green peas, 0.5-1.5%watermelon, 0.5-1.5% honeydew melon, 0.5-1.5% broccoli, 1-2% spinach,0.5-1.5% peppers, 0.5-1.5% snap beans, 0.5-1.5% cantaloupe, 0.4-1.2%cauliflower, 0.5-1.0% mangoes, 0.5-1.0% papaya, 0.3-0.9% celery,0.4-1.2% cucumbers, 0.5-1.0% pineapple, 0.25-0.75% tangerines,0.25-0.75% limes, 0.25-0.75% strawberries, 0.25-0.75% raspberries,0.25-0.75% grapefruit, 0.25-0.75% lemons, 0.25-0.75% cranberries,0.3-0.5% plums, 0.3-0.5% peaches, 0.3-0.5% cherries, 0.3-0.5%blueberries, 0.3-0.5% apricots, 0.1-0.15% dried peas, 0.1-0.15% greatnorthern beans, 0.1-0.15% dried navy beans, 0.1-0.15% dried lentils,0.1-0.15% pinto beans, 0.1-0.15% lima beans, 0.1-0.15% red kidney beans,and 0.1-0.15% black beans.
 6. The composition of claim 5, wherein saidmixture of fruit and vegetable species comprises 18.075% oranges,16.161% tomatoes, 9.595% apples, 14.493% potatoes, 4.373% bananas,3.564% sweet corn, 3.383% grapes, 2.537% lettuce, 1.651% escarole,1.375% brussels sprouts, 1.375% cabbage, 1.329% carrots, 2.017% onions,1.293% green peas, 1.058% watermelon, 1.058% honeydew melon, 0.84%broccoli, 1.651% spinach, 1.087% peppers, 1.061% snap beans, 1.058%cantaloupe, 0.84% cauliflower, 0.732% mangoes, 0.732% papaya, 0.626%celery, 0.814% cucumbers, 0.732% pineapple, 0.555% tangerines, 0.555%limes, 0.437% strawberries, 0.437% raspberries, 0.555% grapefruit,0.555% lemons, 0.437% cranberries, 0.388% plums, 0.388% peaches, 0.388%cherries, 0.437% blueberries, 0.388% apricots, 0.121% dried peas, 0.121%great northern beans, 0.121% dried navy beans, 0.121% dried lentils,0.121% pinto beans, 0.121% lima beans, 0.121% red kidney beans, and0.121% black beans.
 7. The composition of claim 1, wherein the mixturecomprises a polyphenol content of 15-25% hesperetin, 15-25%caffeoylquinic acid, 10-20% quercetin, and 5-15% malvidin.
 8. Thecomposition of claim 7, wherein the mixture comprises 20.6% hesperetin,19.1% caffeoylquinic acid, 15.7% quercetin, and 10.3% malvidin.
 9. Thecomposition of claim 1, wherein the mixture further comprises 1-10%naringenin, 1-10% pelargonidin, 1-5% catechin, and 1-5% procyanidin. 10.The composition of claim 9, wherein the mixture comprises 6.5%naringenin, 5.8% pelargonidin, 4.2% catechin, and 3.1% procyanidin. 11.The composition of claim 1, wherein the mixture further comprises one ormore polyphenols selected from caffeic acid, peonidin, cyanidin,pinoresinol, p-Coumaroyl, luteolin, petunidin, daidzein, genistein,ellagic acid, and gallic acid.
 12. The composition of claim 1, whereinthe composition further comprises one or more of protein, carbohydrates,or fat.
 13. The composition of claim 12, wherein the compositioncomprises 5-15% protein (kcal/kcal), 75-85% carbohydrates (kcal/kcal),and 0-20% fats (kcal/kcal).
 14. The composition of claim 12, wherein thecomposition comprises 10-12% protein (kcal/kcal), 80-83% carbohydrates(kcal/kcal), and 5-10% fat (kcal/kcal).
 15. The composition of claim 12,wherein the composition comprises 11.6% protein (kcal/kcal), 81.2%carbohydrates, and 7.2% fats (kcal/kcal).
 16. The composition of claim2, wherein the dry powder is a freeze-dried powder.
 17. The compositionof claim 2, wherein said composition is a nutritional supplement, food,or beverage.
 18. A method of treating and/or preventing one or moreconditions selected from weight gain, obesity, inflammatory conditions,fatty liver disease, high cholesterol, glucose intolerance, insulinresistance, low gut microbiota diversity, heart disease, andatherosclerosis in a subject, comprising: administering the compositionof claim 1 to said subject.
 19. A method of decreasing fat mass,increasing muscle mass, reducing inflammatory cytokines and/orceramides, reducing tissue inflammation, decreasing cholesterol,improving glucose tolerance, improving immune response, increasing gutmicrobiota diversity, increasing lifespan, improving cognition, and/orimproving bone health in a subject, comprising: administering thecomposition of claim 1 to said subject.
 20. The method of claim 18,wherein said subject is a human.